Antipathogenic benzamide compounds

ABSTRACT

The present invention is related to methods of treating bacterial infections in mammals using antipathogenic benzamide compounds having the formula 
     
       
         
         
             
             
         
       
     
     wherein at least one of the R 1  groups is F, Cl, CN or CF 3  and R 2 , R 3 , Y, Z, m, and n are as defined herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. Nos. 60/342,309, filedDec. 21, 2001, and 60/298,206, filed Jun. 13, 2001; the disclosures ofwhich are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No.N65236-99-1-5427 awarded by the Space and Naval Warfare Systems Command.The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to antipathogenic compounds and methods for theiruse.

2. Description of Related Art

A number of naturally occurring or synthetic compounds bind to doublestranded nucleic acid, especially double stranded DNA (“dsDNA”). Somebind to the major groove, while others bind to the minor groove. Stillothers intercalate between adjacent base pairs. Combination bindingmodes are known, in which a compound has binding interactions with morethan one nucleic acid site.

It has been proposed to use dsDNA binding compounds to regulate theexpression of genes for medical purposes. If a disease is characterizedby the overexpression or undesired expression of a gene (e.g., anoncogene), in principle the disease can be treated by suppressing whollyor partially the gene's expression via the binding of a compound to thegene or a promoter site thereof and interfering with transcription.Infections by pathogens such as fungi, bacteria, and viruses can betreated with compounds that interfere with the expression of genesessential for the pathogen's proliferation. Or, in a diseasecharacterized by non- or under-expression of a beneficial gene, theexpression of the beneficial gene can be up-regulated with a compoundthat binds to the binding site of a repressor, displacing the repressor.

The natural products distamycin and netropsin represent a class ofDNA-binding compounds that has been studied over the years:

Structurally, distamycin and netropsin are heteroaromatic polyamides,having as their core structural motif N-methylpyrrole carboxamideresidues. They bind to the minor groove, their crescent molecular shapesproviding a conformational fit within the groove. The binding occurswith a preference for A,T rich dsDNA tracts.

Many heteroaromatic polyamides have been synthesized elaborating on thedistamycin/netropsin motif, with the objective of enhancing or varyingbiological activity, increasing binding affinity to dsDNA, and/orimproving specificity in base pair sequence recognition. See Bailly etal., Bioconjugate Chemistry 1998, 9 (5), 513-538, and Neidle, Nat. Prod.Rep. 2001, 18, 291-309. The use of synthetic heteroaromatic polyamidesin therapeutics has been proposed, for example, in Dervan et al., U.S.Pat. No. 5,998,140 (1999); Dervan et al., WO 00/15209 (2000); Dervan, WO00/15773 (2000); and Gottesfeld et al., WO 98/35702 (1998).

BRIEF SUMMARY OF THE INVENTION

This invention provides benzamide compounds having the formula

including the pharmaceutically acceptable salts thereof.

Each R¹ is independently H, F, Cl, CN, CF₃, OH, N(R²)₂, OR² or asubstituted or unsubstituted (C₁-C₁₂)alkyl group, or a substituted orunsubstituted (C₁-C₁₂)heteroalkyl group, with the proviso that at leastone R¹ is F, Cl, CN, OCF₃, OCF₂H, or CF₃ (preferably F, Cl, or OCF₂H).Each R² and R³ is independently H, a substituted or unsubstituted(C₁-C₁₂)alkyl group, or a substituted or unsubstituted(C₁-C₁₂)heteroalkyl group.

Each Y is independently a branched or unbranched, substituted orunsubstituted (C₁-C₅)alkylene group or a substituted or unsubstituted,aromatic or heteroaromatic ring system, wherein the ring system has atleast one of a 5- or 6-member aromatic or heteroaromatic ring or fused6, 6 or 6,5 aromatic or heteroaromatic rings, with the proviso that atleast one Y is a substituted or unsubstituted 5-member heteroaromaticring.

Preferably, Y in the moiety —(NR³—Y—CO)— immediately adjacent to

is a 5- or 6-member heteroaromatic ring.

Subscript m is an integer from 1 to 25, inclusive, preferably from 1 to6, more preferably from 2 to 4.

Z is either O or N, with subscript n being 1 if Z is 0 and 2 if Z is N.

Compound (I) has at least one basic group having a pK_(b) of 12 or lessor a quaternized nitrogen group.

Preferably, each moiety —(NR³—Y—CO)— is independently selected from thegroup consisting of:

(a) moieties M¹ having the formula

-   -   wherein one of X¹, X², and X³ is a ring vertex selected from the        group consisting of —O—, —S—, and —NR²—, and the other two of        X¹, X², and X³ are ring vertices selected from the group        consisting of ═N— and ═CR⁴—;

(b) moieties M² having the formula

-   -   wherein x is 0 or 1 and each R¹⁵ is independently H, OH, NH₂, or        F;

(c) moieties M³ having the formula

-   -   wherein each L is independently a divalent moiety separating        —NH— and —(C═O)— by 3 or 4 atoms; and

(d) moieties M⁴ having the formula

with the proviso that at least one moiety —(NR³—Y—CO)— is a moiety M¹.

In the preceding formulae M¹ to M⁴, R² and R³ are as previously definedand each R⁴ is independently H, F, Cl, Br, I, CN, OH, NO₂, NH₂, asubstituted or unsubstituted (C₁-C₁₂)alkyl group, or a substituted orunsubstituted (C₁-C₁₂)heteroalkyl group.

Preferably, the moiety —(NR³—Y—CO)— immediately adjacent to the residue

is a moiety M¹.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIGS. 1 a-1 c, 2 a-2 b, and 3 illustrate compounds according to thisinvention.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations and Definitions

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,cyclopropylmethyl, homologs and isomers of, for example, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group isone having one or more double bonds or triple bonds. Examples ofunsaturated alkyl groups include vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified by—CH₂CH₂CH₂CH₂—. Typically, an alkyl (or alkylene) group will have from 1to 24 carbon atoms, with those groups having 10 or fewer carbon atomsbeing preferred in the present invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingsix or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from one to three heteroatoms selectedfrom the group consisting of O, N, Si and S, and wherein the nitrogenand sulfur atoms may optionally be oxidized and the nitrogen heteroatommay optionally be quaternized. The heteroatom(s) O, N and S may beplaced at any interior position of the heteroalkyl group. The heteroatomSi may be placed at any position of the heteroalkyl group, including theposition at which the alkyl group is attached to the remainder of themolecule. Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified by—CH₂—CH₂—S—CH₂CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is meant to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,typically aromatic, hydrocarbon substituent which can be a single ringor multiple rings (up to three rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from zero to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) are meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl, heteroalkyl, aryl, and heteroalkyl radicals(including those groups often referred to as alkylene, alkenyl,heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl,cycloalkenyl, and heterocycloalkenyl) can be a variety of groupsselected from: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂ in a numberranging from zero to (2 m′+1), where m′ is the total number of carbonatoms in such radical. R′, R″ and R′″ each independently refer tohydrogen, unsubstituted (C₁-C₈)alkyl and heteroalkyl, unsubstitutedaryl, aryl substituted with 1-3 halogens, unsubstituted alkyl, alkoxy orthioalkoxy groups, or aryl-(C₁-C₄)alkyl groups. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 5-, 6-, or 7-membered ring. For example, —NR′R″is meant to include 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups such as haloalkyl (e.g.,—CF₃ and —CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, andthe like). Preferably, the substituted alkyl and heteroalkyl groups havefrom 1 to 4 substituents, more preferably 1, 2 or 3 substituents.Exceptions are those perhalo alkyl groups (e.g., pentafluoroethyl andthe like) which are also preferred and contemplated by the presentinvention.

Similarly, substituents for the aryl and heteroaryl groups are variedand are selected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN,—NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)₂R′,—NR′—C(O)NR″R′″, —S(O)₂R′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′,—S(O)R′, —S(O)₂NR′R″, —N₃, —CH(Ph)₂, perfluoro(C₁-C₄)alkoxy, andperfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total numberof open valences on the aromatic ring system; and where R′, R″ and R′″are independently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl,and (unsubstituted aryl)oxy-(C₁-C₄)alkyl.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, —O—, —CH₂—or a single bond, and q is an integer of from 0 to 2. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula-A-(CH₂)_(r)—B—, wherein A and B are independently —CH₂—, —O—, —NH—,—S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integerof from 1 to 3. One of the single bonds of the new ring so formed mayoptionally be replaced with a double bond. Alternatively, two of thesubstituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituent R′ in —NR′— and —S(O)₂NR′— is selected from hydrogen orunsubstituted (C₁-C₆) alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, ascorbic, propionic, isobutyric, maleic, malonic, lactic, malic,glutamic, benzoic, succinic, suberic, fumaric, mandelic, phthalic,benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic,lactobionic, and the like. Also included are salts of amino acids suchas arginate and the like, and salts of organic acids like glucuronic orgalactunoric acids and the like (see, for example, Berge, S. M., et al,“Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66,1-19). Certain specific compounds of the present invention contain bothbasic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (chiral centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are all intended to beencompassed within the scope of the present invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

In the discussions below, reference is made to dsDNA as the nucleicacid, but it is to be understood that the invention is not limited todsDNA and is applicable to other nucleic acids, i.e., ribonucleic acid.

Compounds

Compounds (I) of this invention are poly- or oligoamides having abenzamide unit and heteroaromatic carboxamide units.

Compounds (I) are believed to be DNA-binding compounds, which bind tothe minor groove of dsDNA. Different polyamide-dsDNA binding modes arepossible. In the simplest mode, often referred to as the 1:1 bindingmode, a single polyamide molecule fits in the channel formed by theminor groove. In what is referred to as the 2:1 binding mode, twopolyamide molecules fit side-by-side in the minor groove, preferablyaligned in an antiparallel manner (i.e., with one polyamide beingaligned N-to-C and the other polyamide being aligned C-to-N, where “C”and “N” refer to the carboxy and amino termini, respectively of thepolyamides). Lastly, in what is referred to as a “hairpin” binding mode,a single polyamide molecule that has a more or less centrally positionedflexible moiety (i.e., a moiety M³, as discussed in greater detailhereinbelow) folds around itself to adopt a hairpin conformation when itis bound to the minor groove, so that a first portion of the polyamideat one side of the hairpin turn is adjacent to a second portion of thepolyamide at the other side of the hairpin turn.

In formula (I)

the residue

preferably is selected from the group consisting of

Moieties M¹, described by formulae IIa and IIb

provide preferred heteroaromatic polyamide building blocks. Moieties M¹are 5-membered ring heteroaromatic moieties, the selection of X¹, X²,and X³ determining the type of heteroaromatic ring. Exemplaryheteroaromatic rings include imidazole, pyrrole, pyrazole, furan,isothiazole, oxazole, isoxazole, thiazole, furazan, 1,2,3-thiadiazole,1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3-triazole,1,2,4-triazole, 1,3,4-oxadiazole, 1,2,4-oxadiazole, and thiophene.

The circle in the five-membered rings of formulae IIa and IIb is meantto indicate the presence of two double bonds, which, in someembodiments, can move within the ring.

Preferred moieties M¹ are IIc (hereinafter “Py”), formally derived from1-methyl-4-aminopyrrole-2-carboxylic acid, IId (hereinafter “Hp”),formally derived from 1-methyl-3-hydroxy-4-aminopyrrole-2-carboxylicacid, and IIe (hereinafter “Im”), formally derived from1-methyl-4-aminoimidazole-2 carboxylic acid:

It has been shown by Dervan and co-workers (see, e.g., Dervan, U.S. Pat.No. 6,143,901 (2000); Dervan et al., WO 98/37066 (1998); White et al.,Nature 391, 468 (1998); White et al., Chem. Biol. 1997, 4, 569) that, ina 2:1 binding mode to dsDNA, moieties Py, Im, and Hp moieties can beused to recognize specific dsDNA base pairs, giving rise to a set of“pairing rules” correlating heteroaromatic moiety pairs and DNA basepairs. These pairing rules are summarized below:

Heteroaromatic Pair dsDNA Base Pair(s) Recognized Im/Py G/C Py/Im C/GPy/Py A/T, T/A (degenerate) Hp/Py T/A Py/Hp A/TSuch recognition can lead to sequence-specific dsDNA binding, enablingthe design of compounds (I) that target predetermined DNA base pairsequences, for example, a specific promoter site or a sequencecharacteristic of a gene.

Optionally, compound (I) can include one or more moieties M²

A moiety M² can function as a “spacer” for adjusting the positioning ofthe heteroaromatic moieties M¹ or M⁴ relative to the dsDNA base pairs atthe binding site. As a compound (I) binds in the minor groove, thealignment of heteroaromatic moieties M¹ and M⁴ with the DNA base pairswith which they to interact of optimal binding or sequence recognitionmay drift as the number of heteroaromatic moieties M¹ and M⁴ increases.Alternatively, incorporation of a moiety M² adds flexibility to compound(I), allowing its curvature to more accurately match that of the minorgroove. The incorporation of one or more flexible moieties M² relaxesthe curvature of the compound backbone, permitting larger compounds (I)to bind to longer sequences of DNA. In some preferred embodiments amoiety M² is present for every 4 to 5 heteroaromatic moieties M¹ or M⁴,more preferably interrupting long sequences of M¹ and/or M⁴ groups.

Preferred moieties M² are those corresponding to glycine (x=0 in formulaIII, depicted as Ma below) and β-alanine (n=1 and R¹⁵═H in formula III;depicted as Mb below, hereinafter “β”), with the latter being especiallypreferred.

Moieties M² in which x=1 and R¹⁵═OH, NH₂, or F can be used to alter thebinding affinity and specificity (relative to β-alanine), as disclosedin Floreancig et al., J. Am. Chem. Soc., 2000, 122, 6342; the disclosureof which is incorporated herein by reference.

When present in compound (I), optional moieties M³ (formula IV)

have a group L providing a spacer of 3 to 4 atoms between —NH— and—C(═O)— and can be used to introduce a hairpin turn into compound (I).See Mrksich et al., J. Am. Chem. Soc. 1994, 116, 7983. Exemplarymoieties M³ include:

Moieties IVa (hereinafter “y”), corresponding to γ-aminobutyric acid,and IVc, corresponding to 2,4-diaminobutyric acid, are preferred.Selecting one enantiomer or the other of moieties M³ that are chiralallows stereochemical control of the binding of polyamides to the minorgroove, for example as disclosed in Baird et al., WO 98/45284 (1998) inrespect of R-2,4-diaminobutyric acid and S-2,4-diaminobutyric acid(corresponding to R-IVc and S-IVc, respectively).

Yet another class of moieties M³ is represented by the formula

While the group L preferably provides a 3-atom separation between the—NH— and the —(C═O)—, a 4-atom separation is also permissible, asillustrated by a 5-aminovaleric acid residue (i.e., L equals —(CH₂)₄—):

L can have pendant groups, which serve to enhance solubility or functionas attachment points for other groups (e.g., IVc, IVd, IVg, IVh, IVk,IVl). The 3 to 4 atoms can be part of a larger group, which providesconformational rigidity (e.g., IVj). The 3 to 4 atoms can comprisecarbon atoms only or it can include heteroatoms (e.g., IVb, IVe, IVi).

Moieties M⁴ optionally can be used to introduce a aromatic orheteroaromatic residues other than M¹ into compound (I), in particulararyl-benzimidazole, pyridinyl carboxamide, or benzamide residues.

The group Z(R²)_(n) can be viewed as a terminal group, forming an amideor ester cap at the C-terminus of compound (I). In the case of Z═N, thetwo groups R² can be linked to each other to form a cyclic structure. Agroup Z(R²)_(n) can contain a basic group (as defined hereinbelow).Examples of suitable groups Z(R²)_(n) containing a basic group include:

Examples of suitable groups Z(R²)_(n) not containing a basic groupinclude:

In the foregoing formulae, r is an integer ranging from 2 to 8,inclusive (preferably 2 to 6), and each R²⁵ is independently H, CH₃,CH₂CH₃, CH₂CH₂CH₃, or CH(CH₃)₂.

Compounds of this invention having such groups Z(R²)_(n) preferably havea basic group having a pK_(b) of 12 or less or a quaternized nitrogengroup located in a moiety M¹ or M⁴ or in the N-terminal benzamide group.

The classification of the 5-amino-3-methylisothiazole group as a“nonbasic” Z(R²)_(n) group is somewhat arbitrary, as its pK_(b) ismarginal, normally around 12-13 (i.e., pK_(a) 1-2) and depending on themolecular structure of the entire compound, it may qualify or not as abasic group as such is defined herein. Preferably, where a5-amino-3-methylisothiazole is present, the compound has a basic groupelsewhere in the molecule, for example pendant from a moiety M¹ or M⁴,as exemplified by compounds C-21 to C-25, D-1, D-6, D-8, D-15 to D-24,D-28 to D-29, and E-7, infra.

The preceding illustrative formulae of basic and nonbasic groupsZ(R²)_(n) have been drawn with Z as N and n as 2 for convenience. Thoseskilled in the art will appreciate that in these formulae the residueNR²⁵ can be replaced with 0 to give the corresponding groups Z(R²)_(a)in which Z is O and n is 1. Where Z is O, preferably the adjacent moietyY is Py.

As used herein with reference to groups R² and R³, “substituted orunsubstituted (C₁-C₁₂)alkyl group, or a substituted or unsubstituted(C₁-C₁₂)heteroalkyl group” includes not only conventional alkyl orcycloalkyl groups such as methyl, ethyl, propyl, isopropyl, butyl,s-butyl, isobutyl, t-butyl, cyclopentyl, cyclohexyl, and pentyl, butalso unsaturated C₁ to C₁₂ groups, having for example aromatic, alkenyl,or alkynyl groups (e.g., phenyl, benzyl, vinyl, cyclohexenyl, etc.). Oneor more backbone carbons can be replaced by heteroatoms. There may bepresent functionalities such as hydroxy; oxo (═O); primary, secondary,or tertiary amine (e.g., —NH₂, —NH(CH₃), —N(CH₃)₂); quaternary ammonium(e.g., —N(CH₃)₃ ⁺); alkoxy (e.g., methoxy, ethoxy); acyl (e.g.,—C(═O)CH₃); amide (e.g., —NHC(═O)CH₃); thiol; thioether (e.g., —SCH₃);sulfoxide; sulfonamide (e.g., —SO₂NHCH₃); halogen (e.g., F, Cl); nitro;and the like. Exemplary specific R¹ and R² groups include methyl,trifluoromethyl, ethyl, acetyl, methoxy, methoxyethyl, ethoxyethyl,aminoethyl, hydroxyethyl, propyl, hydroxypropyl, cyclopropyl, isopropyl,3-(dimethylamino)propyl, butyl, s-butyl, isobutyl, t-butyl, pentyl,cyclopentyl, vinyl, allyl, ethynyl, propynyl, and the like.

Compound (I) has a basic group having a pK_(b) of 12 or less or aquaternized nitrogen group. (Or, stated conversely, the conjugate acidof the basic group has a pK_(a) greater than 2 (pK_(a)=14-pK_(b)).)Preferably, the pK_(b) is less than 10, more preferably less than 5. ApK_(b) of less than 12 ensures that compound (I) is protonated under theconditions in which it interacts with a nucleic acid. Preferably thebasic group is a nitrogenous group, for example an amine, an amidine, aguanidine, a pyridine, a pyridazine, a pyrazine, a pyrimidine, animidazole, or an aniline. Primary, secondary, or tertiary aliphaticamines are preferred. Exemplary quaternized nitrogen groups includealkyl pyridinium and tetraalkyl ammonium groups such as:

A basic/quaternized nitrogen group may improve the transport of thecompounds of this invention across cellular and nuclear membranes, toreach dsDNA in the nucleus. See Rothbard et al., WO 98/52614 (1998),which discloses that guanidine or amidino side chain moieties enhancetransport across biological membranes. Another possible benefit isimproved binding affinity to the nucleic acid, via ionic interactionswith backbone phosphate groups. See Baird and Dervan, WO 98/37087 (1998)and Bruice et al., U.S. Pat. No. 5,698,674 (1997). Lastly, theprotonated basic or quaternized nitrogen group increases solubility.

Preferably, the basic or quaternized nitrogen group is present withinthe C-terminal group Z(R²)_(n), but it may be present elsewhere in themolecule, for example as part of a group R¹ or R² in M¹ or M⁴. Or,multiple basic or quaternized nitrogen groups may be present, atdifferent locations on compound (I).

In a preferred embodiment, compound (I) is according to formula (Ia):

wherein M², M³, R¹, R², Z and n have the same meanings as previouslyassigned; each A is independently M¹ or M⁴; each of a, c, e, g and h isan integer independently from 0 to 4, inclusive; and each of b, d, and fis independently 0 or 1, with the proviso that at least one A is M¹. Thesum of a, c, e, and g is preferably at least 2, more preferably at least3. In another preferred embodiment, each of b, d, and f is 0.

In another preferred embodiment, compound (I) is according to formula(Ib):

wherein X¹, X², X³, R¹, R², R³, Z, and n have the meanings previouslyassigned and i is an integer from 1 to 4, inclusive (preferably 2 or 3).In a preferred subgenus, the first 5-member heterocyclic ring (readingfrom left to right) is an unsubstituted pyrrole while subsequent5-member heterocyclic rings are N-methylpyrroles, i.e., according toformula (Ic):

R¹, R², i, Z and n have the meanings assigned previously. Examples ofcompounds according to formula (Ic) are provided in Table A:

TABLE A Illustrative Compounds (Ic) Compound Ref.

i

 A-1

3

 A-2 Same 4 Same  A-3

3 Same  A-4 Same 3

 A-5 Same 3

 A-6 Same 3

 A-7 Same 3

 A-8 Same 3

 A-9 Same 3

A-10 Same 3

A-11 Same 3

A-12 Same 3

A-13

3

A-14 Same 3

A-15 Same 3

A-16 Same 3

A-17 Same 2

A-18 Same 2

A-19 Same 2

A-20 Same 2

A-21 Same 2

A-22 Same 2

A-23 Same 2

A-24 Same 3

A-25 Same 2

A-26

3

A-27 Same 4

A-28 Same 3

A-29 Same 3

A-30

3

A-31

3 Same A-32

3 Same A-33

3 Same A-34

3 Same A-35

3 Same

In another preferred subgenus of compounds according to formula (Ib),each of the 5-member heterocylic rings is N-methylpyrrole, i.e.,according to formula (Id):

R¹, R², i, Z and n have the meanings assigned previously. Examples ofcompounds according to formula Id are provided in Table B

TABLE B Illustrative Compounds (Id) Compound Ref.

i

 B-1

3

 B-2 Same 2 Same  B-3 Same 3

 B-4 Same 3

 B-5 Same 3

 B-6 Same 3

 B-7 Same 3

 B-8 Same 3

 B-9 Same 3

B-10 Same 3

B-11 Same 2

B-12 Same 2

B-13 Same 2

B-14 Same 2

B-15

2

B-16 Same 2

B-17 Same 3

B-18 Same 3

B-19 Same 3

B-20 Same 3

B-21 Same 3

B-22 Same 3

B-23 Same 3

B-24 Same 3

B-25 Same 3

B-26 Same 3

B-27 Same 3

B-28 Same 3

B-29

3

B-30 Same 3

B-31

3

B-32

3 Same B-33 Same 2

B-34 Same 3 Same B-35 Same 4 Same B-36 Same 2

B-37 Same 4 Same B-38

3 Same B-39

3 Same B-40 Same 2

B-41 Same 3 Same B-42 Same 4 Same B-43 Same 2

B-44 Same 4

B-45

3

B-46

3 Same B-47

3 Same B-48 Same 2 Same B-49 Same 3

B-50 Same 3

B-51

3

B-52

3

B-53

3 Same B-54 Same 2 Same B-55

3 Same B-56

3 Same B-57 Same 2 Same B-58 Same 3

B-59

3

B-60

3 Same B-61

3 Same B-62

3 Same B-63

3

B-64

3

B-65 Same 3

B-66

3 Same B-67

3

B-68 Same 3

B-69 Same 2

B-70

3 Same B-71

3

B-72

3 Same B-73

3 Same B-74

3 Same B-75

3 Same B-76

3 Same B-77

3 Same B-78

3 Same B-79

3 Same B-80

3 Same B-81

3 Same B-82

3

B-83

3

B-84

3

B-85 Same 3

B-86 Same 3

In another preferred subgenus of compounds within formula (Ib), each ofthe 5-member heterocycles is a pyrrole, as given by formula (Ie)

where R¹, R², i, Z, and n are as previously defined, with the provisothat at least one R² bonded to a pyrrole nitrogen is other than CH₃.Exemplary such compounds are shown in Table C. (In the column headed“R^(2a), R^(2b), R^(2c), and R^(2d),” R^(2a) represents the group R²attached to the first pyrrole ring (reading from left to right), R^(2b)represents the group R² attached to the second pyrrole ring, and soforth. To illustrate, the first compound listed in Table C(C-1) is

(R^(2d) is “n/a” for “not applicable,” because there is no fourthpyrrole ring in this instance.)

TABLE C Illustrative Compounds Ie Compound Ref.

i R^(2a), R^(2b), R^(2c), and R^(2d)

 C-1

3 R^(2a) = R^(2b) = CH₃, R^(2c) = H, R^(2d) = n/a

 C-2 Same 3 R^(2a) = CH₃, Same R^(2b) = H, R^(2c) = CH₃, R^(2d) = n/a C-3 Same 3 R^(2a) = R^(2b) = H, Same R^(2c) = CH₃, R^(2d) = n/a  C-4Same 3 R^(2a) = CH₃, Same R^(2b) = (CH₂)₃OH, R^(2c) = CH₃, R^(2d) = n/a C-5 Same 3 R^(2a) = H, Same R^(2b) = (CH₂)₃OH, R^(2c) = CH₃, R^(2d) =n/a  C-6 Same 3 R^(2a) = (CH₂)₃OH, Same R^(2b) = R^(2c) = CH₃, R^(2d) =n/a  C-7 Same 3 R^(2a) = CH₂OCH₃, Same R^(2b) = R^(2c) = CH₃, R^(2d) =n/a  C-8

3 R^(2a) = H, R^(2b) = CH₃, R^(2c) = H, R^(2d) = n/a Same  C-9 Same 3R^(2a) = H, R^(2b) = CH₃, R^(2c) = (CH₂)₃OH, R^(2d) = n/a

C-10 Same 3 R^(2a) = CH₃, R^(2b) = H, R^(2c) = CH₃, R^(2d) = n/a

C-11 Same 3 R^(2a) = CH₃, Same R^(2b) = H, R^(2c) = CH₃, R^(2d) = n/aC-12 Same 3 R^(2a) = H, Same R^(2b) = CH₂CH₃, R^(2c) = CH₃, R^(2d) = n/aC-13 Same 3

Same C-14

3 R^(2a) = H, R^(2b) = (CH₂)₃OH, R^(2c) = CH₃, R^(2d) = n/a

C-15 Same 3 R^(2a) = H, Same R^(2b) = (CH₂)₃OH, R^(2c) = CH₃, R^(2d) =n/a C-16 Same 3 R^(2a) = (CH₂)₃OH, Same R^(2b) = R^(2c) = CH₃, R^(2d) =n/a C-17 Same 3 R^(2a) = CH₂OCH₃, Same R^(2b) = R^(2c) = CH₃, R^(2d) =n/a C-18 Same 3 R^(2a) = CH₃, Same R^(2b) = (CH₂)₃Cl, R^(2c) = CH₃,R^(2d) = n/a C-19 Same 3 R^(2a) = H, Same R^(2b) = (CH₂)₃Cl, R^(2c) =CH₃, R^(2d) = n/a C-20

4 R^(2a) = CH₃, R^(2b) = (CH₂)₃SCH₂CH₃, R^(2c) = R^(2d) = CH₃ Same C-21

2 R^(2a) = CH₃ R^(2b) = H R^(2c) = R^(2d) = n/a

C-22

2 R^(2a) = CH₃ R^(2b) = H R^(2c) = R^(2d) = n/a Same C-23

2 R^(2a) = CH₃ R^(2b) = H R^(2c) = R^(2d) = n/a Same C-24

2 R^(2a) = CH₃ R^(2b) = H R^(2c) = R^(2d) = n/a Same C-25

2 R^(2a) = CH₃ R^(2b) = H R^(2c) = R^(2d) = n/a Same

In a preferred type of compounds (Ie), at least one R² bonded to apyrrole ring has a basic group having a pK_(b) of 12 or less or aquaternized nitrogen group. Exemplary such compounds are shown in TableD, with the column headed “R^(2a), R^(2b), R^(2c), and R^(2d)” construedin the same manner as in Table C.

TABLE D Illustrative Compounds Ie Compound Ref.

i R^(2a), R^(2b), R^(2c), and R^(2d)

 D-1

2

 D-2 Same 3 R^(2a) = R^(2b) = CH₃, R^(2c) = (CH₂)₃N(CH₃)₂, R^(2d) = n/a

 D-3 Same 2 R^(2a) = CH₃, R^(2b) = (CH₂)₃N(CH₃)₂, R^(2c) = R^(2d) = n/a

 D-4 Same 2

Same  D-5 Same 3

 D-6 Same 2 R^(2a) = CH₃, R^(2b) = (CH₂)₃N⁺(CH₃)₃, R^(2c) = R^(2d) = n/a

 D-7 Same 3 R^(2a) = —CH₃, R^(2b) = (CH₂)₃N(CH₃)₂, R^(2c) = CH₃, R^(2d)= n/a

 D-8

2

 D-9 Same 2

Same D-10 Same 2

Same D-11

2 R^(2a) = H, R^(2b) = (CH₂)₃N(CH₃)₂, R^(2c) = R^(2d) = n/a

D-12 Same 2 R^(2a) = CH₃, Same R^(2b) = (CH₂)₃N(CH₃)₂, R^(2c) = R^(2d) =n/a D-13 Same 3

D-14 Same 3

Same D-15 Same 2

D-16 Same 2

Same D-17 Same 2

Same D-18 Same 2

Same D-19 Same 2

Same D-20 Same 2

Same D-21 Same 2 R^(2a) = (CH₂)₃N(CH₂CH₂OH)₂, Same R^(2b) = H, R^(2c) =R^(2d) = n/a D-22 Same 2 R^(2a) = CH₃, Same R^(2b) = (CH₂)₃N⁺(CH₃)₃,R^(2c) = R^(2d) = n/a D-23

2 R^(2a) = H, R^(2b) = (CH₂)₃N⁺(CH₃)₃, R^(2c) = R^(2d) = n/a

D-24 Same 2

Same D-25 Same 3 R^(2a) = CH₃, R^(2b) = (CH₂)₃N(CH₃)₂, R^(2c) = CH₃,R^(2d) = n/a

D-26 Same 3 R^(2a) = H, Same R^(2b) = (CH₂)₃N(CH₃)₂, R^(2c) = CH₃,R^(2d) = n/a D-27

3

D-28 Same 2

D-29

2

Same

However, the moieties

in formula (Ib) need not all be pyrrole carboxamides, as was in theinstance of formulae (Ic), (Id), and (Ie). They can be other thanpyrrole, as illustrated in FIGS. 1 a to 1 c.

Preferred non-pyrrole 5-member ring heterocycles include:

In yet another preferred embodiment, compounds (I) of this invention areaccording to formula (If):

wherein M¹, M⁴, R¹, R², Z, and n are as previously defined and each k isindependently an integer from 0 to 4, inclusive, with the proviso thatat least one k is not 0. Illustrative compounds (If) are shown in FIGS.2 a through 2 b.

Compounds (Ia), (Ib), (Ic), (Id), (Ie), and (If) have a basic grouphaving a pK_(b) of 12 or less or a quaternized nitrogen group.

While it is normally preferred that the group R³ in —(NR³—Y—CO)— is H,such need not invariably be the case. R³ can be alkyl or alkylamino oralkylammonium, i.e., possessing a basic group having a pK_(b) of 12 orless or a quaternary nitrogen group. Examples of such compounds areshown in FIG. 3.

Compounds (I) can be conjugated or linked to another nucleic acidbinding compound. The conjugated nucleic acid binding compounds can betwo identical or different compounds (I), or one compound (I) and adifferent class of nucleic acid binder, for example an intercalator, atriple helix former, a binder to the phosphate backbone, a major groovebinder, another type of minor groove binder, and the like. A preferredsite for forming the conjugating link is an amino, hydroxy, or thiolfunctionality in a group L in moiety M², which can be acylated oralkylated. The preparation of tandem linked nucleic acid bindingpolyamides in this manner is disclosed in Baird et al., WO 98/45284(1998), the disclosure of which is incorporated herein by reference.

Compounds (I) also can be conjugated to other moieties, such as,peptides, proteins, transport agents, fluorophores or other reportergroups, and the like.

Compounds (I) preferably bind to dsDNA with high affinity, meaning anequilibrium association constant of at least 10³ M⁻¹, more preferably atleast 10⁶ M⁻¹, and most preferably at least 10⁹ M⁻¹. The measurement ofbinding affinities by quantitative DNase I footprinting is disclosed inDervan, WO 98/50582 (1998), and Trauger et al., Nature 382, 559 (8 Aug.1996); the disclosures of which are incorporated herein by reference.

Compounds of this invention can be used to form complexes with dsDNA,for the purpose of recognizing and/or isolating dsDNA strands containingparticular base-pair sequences, for example for analytical or diagnosticpurposes. Thus, in another aspect of this invention there is provided acomplex between dsDNA and compound of this invention. In cellularsystems or in living organisms, they can modulate the expression of agene by binding to the gene or a promoter or repressor region thereof.Such modulation may be useful for therapeutic or research purposes.

Additionally, compounds of this invention have been found to haveanti-bacterial and/or antifungal properties and therefore may be usedfor combating (i.e., preventing and/or treating) infections ineukaryotic organisms. Other pathogens against which compounds of thisinvention can be useful include protozoa and viruses. For humananti-infective applications, an effective amount of a compound of thisinvention is used, optionally in combination with a pharmaceuticallyacceptable carrier. The composition may be dry, or it may be a solution.Treatment may be reactive, for combating an existing infection, orprophylactic, for preventing infection in an organism susceptible toinfection. Preferably, compounds of this invention are used to treatinfections by drug-resistant strains of bacteria, for example MRSA(methycillin resistant S. aureus), MRSE (methycillin resistant S.epidermidis), PRSP (penicillin resistant S. pneumoniae) or VRE(vancomycin resistant Enterococci). By “drug-resistant” it is meant thatthe bacteria are resistant to treatment with conventional antibiotics.

Host organisms that can be treated include eukaryotic organisms, inparticular plants and animals. The plant may be an agriculturallyimportant crop, such as wheat, rice, corn, soybean, sorghum, andalfalfa. Animals of interest include mammals such as bovines, canines,equines, felines, ovines, porcines, and primates (including humans).Thusly, in another aspect of this invention, there is provided a methodfor treating a bacterial infection—particularly an infection byGram-positive bacteria—comprising administering to a patient in need ofsuch treatment an effective amount of compound (I). Compounds of thisinvention can be used in the preparation of a medicament for treating abacterial infection in a mammal. The compounds may be administeredorally, topically, or parenterally (e.g., intravenously, subcutaneously,intraperitoneally, transdermally).

While not wishing to be bound by any particular theory, it is believedthat the compounds of this invention derive their biological activityfrom their ability to bind to dsDNA.

Compounds I can be synthesized by solid phase techniques from thecorresponding amino acids or their derivatives, for instance IIc′, IId′,and Ile′ for the synthesis of the Py, Hp, and Im building blocks,respectively.

In solid phase synthesis, a polyamide is synthesized on a resin such asBoc-glycine-PAM-resin or Boc-β-alanine-PAM-resin, with moieties Y beingadded in series of steps involving amino-protected and carboxy-activatedmonomers, as taught in Dervan et al., U.S. Pat. No. 6,090,947 (2000)(the “'947 patent”); Baird et al., WO 98/37066 (1998); Baird et al., WO98/37067 (1998); and Dervan et al., WO 98/49142 (1998); Baird et al.,U.S. Provisional Appl'n No. 60/286,454, filed Apr. 26, 2001 (the “'454application”); McMinn, U.S. Provisional Appl'n No. 60/298,206, filedJun. 13, 2001 (the “'206 application”); Ge et al., U.S. Appl'n No.09/808,729, filed Mar. 14, 2001 (the “'729 application”); Kelly et al.,Proc. Nat'l Acad. Sci. USA, July 1996, 93, 6981 (“Kelly”); and Wade etal., J. Am. Chem. Soc., 1992, 114, 8783 (“Wade”); the disclosures ofwhich are incorporated herein by reference.

The practice of this invention may be further understood by reference tothe following examples, which are provided by way of illustration andnot of limitation.

In Vitro Biological Activity

In vitro biological activity data were collected for a variety ofmicroorganisms, including Bacillus cereus (ATCC 11778), Staphylococcusaureus (ATCC 27660, a methicillin resistant strain (MRSA); ATCC 13709, amethicillin sensitive strain (MSSA)); Streptococcus pneumoniae (ATCC51422, a penicillin resistant strain (PRSP)), Enterococcus faecium (ATCC51559, a vancomycin resistant strain (VRE)), and Staphylococcusepidermidis (ATCC 12228). Additionally, antifungal activity data werecollected for Candida albicans (ATCC 38247).

Compounds according to this invention were screened for their in vitroactivities against selected species of bacteria and fungi. The minimalinhibition concentration (MIC) of these compounds was determined usingthe National Committee for Clinical Laboratory Standards (NCCLS) brothmicrodilution assay in microtiter plates, as set forth in: (1) theguidelines of the National Committee for Clinical Laboratory Standards(NCCLS) Document M7-A4 (NCCLS, 1997); (2) the guidelines of the NationalCommittee for Clinical Laboratory Standards (NCCLS) Document M11-A4(NCCLS, 1997); and (3) the guidelines and reference method of theNational Committee for Clinical Laboratory Standards (NCCLS) DocumentM27-T (NCCLS, 1995). For antifungal assays, the method recommended inMurray, P R., 1995 Manual of Clinical Microbiology (ASM Press,Washington, D.C.), was employed.

Preferably, compounds of this invention have an MIC of 4 or less againstat least one strain of drug resistant bacteria. The results arepresented in Table E below, which is keyed as follows:

Key to organisms tested against:

-   A=B. cereus ATCC 11778-   B=C. albicans ATCC 38247-   C=E. faeealis ATCC 29212-   D=E. faecium ATCC 51559-   E=S. aureus ATCC 13709-   F=S. aureus ATCC 27660-   G=S. epidermldls ATCC 12228-   H=S. pneumoniae ATCC 49619-   I=S. pneumoniae ATCC 51422    Key to activity:-   +++=MIC<4-   ++=4 ≦MIC<12-   +=12 ≦MIC<32-   ND=not determined-   >32 =preliminary data indicates MIC greater than 32

TABLE E In Vitro Biological Activity Cpd. Organism (Minimum InhibitoryConcentration (MIC), μg/mL) Ref. A B C D E F G H I A-1 +++ >32 +++ ND+++ ++ ND +++ ND A-2 +++ >32 ++ + ND + + ND ND A-3 +++ + +++ +++ +++ ++++++ +++ +++ A-4 +++ ++ + +++ +++ +++ +++ +++ +++ A-5 + >32 +++ ND ++++++ ND ++ ND A-6 +++ >32 +++ +++ +++ +++ +++ +++ +++ A-7 +++ >32 +++ ++++++ +++ +++ +++ +++ A-8 +++ + +++ +++ +++ +++ +++ +++ +++ A-9 +++ + ++++++ +++ +++ +++ + +++ A-10 +++ ++ ++ ND +++ +++ ND +++ ND A-11 + + + ND++ + ND + ND A-12 +++ >32 +++ +++ +++ + +++ +++ +++ A-13 +++ +++ ++ ++++ +++ +++ +++ +++ A-14 +++ + +++ ND +++ +++ ND +++ ND A-15 +++ + +++ND +++ +++ ND +++ ND A-16 +++ +++ +++ +++ +++ +++ +++ +++ +++ A-17 + >32+++ ND ++ ++ ND +++ ND A-18 +++ >32 +++ +++ +++ +++ +++ +++ +++A-19 + >32 +++ ND ++ +++ ND +++ ND A-20 + >32 +++ ND ++ ++ ND +++ NDA-21 +++ >32 +++ +++ +++ +++ +++ +++ +++ A-22 +++ >32 +++ +++ +++ ++++++ +++ +++ A-23 + >32 + ND + + ND +++ ND A-24 +++ >32 ++ ND >32 +++ ND+++ ND A-25 ++ >32 +++ ND +++ +++ ND +++ ND A-26 +++ ++ +++ ND +++ +++ND +++ ND A-27 +++ >32 ++ ND + + ND +++ ND A-28 +++ +++ ++ + +++ +++ ++++++ +++ A-29 +++ >32 + ND +++ ++ ND +++ ND A-30 +++ >32 +++ ND +++ +++ND +++ ND A-34 +++ + ++ ND ND ND ND + ND A-35 +++ >32 +++ ND +++ +++ ND+++ ND B-1 +++ >32 +++ +++ +++ +++ +++ +++ +++ B-2 ++ >32 +++ ND ND NDND +++ ND B-3 +++ >32 +++ + +++ +++ +++ +++ +++ B-4 ++ >32 ++ ND ++ ++ND ++ ND B-5 +++ >32 +++ ND +++ +++ ND +++ ND B-6 +++ >32 +++ +++ ++++++ +++ +++ +++ B-7 +++ >32 +++ ND +++ +++ ND +++ ND B-8 ++ >32 ++ ND ++++ ND ++ ND B-9 +++ >32 +++ ND +++ +++ ND +++ ND B-10 +++ >32 +++ ND + +ND +++ ND B-11 +++ >32 +++ +++ +++ +++ +++ +++ +++ B-12 +++ >32 +++ ND++ +++ ND +++ ND B-13 + >32 ++ ND ++ + ND +++ ND B-14 ++ >32 +++ ND ++++++ ND +++ ND B-15 >32 >32 >32 ND >32 >32 ND + ND B-16 ++ >32 +++ ND ++++ ND +++ ND B-17 +++ >32 + ND ++ ++ ND ++ ND B-18 +++ ++ +++ +++ ++++++ +++ +++ +++ B-19 +++ >32 +++ ND +++ +++ ND +++ ND B-20 +++ >32 + ND+++ ++ ND +++ ND B-21 + + ++ ND + ++ ND ++ ND B-22 +++ >32 +++ +++ ++++++ +++ +++ +++ B-23 +++ ++ +++ ND +++ +++ ND +++ ND B-24 +++ >32 ++++++ +++ +++ +++ +++ +++ B-25 + >32 + ND + + ND +++ NDB-26 >32 >32 >32 >32 >32 >32 >32 >32 >32 B-27 +++ >32 +++ +++ +++ ++++++ +++ +++ B-28 + >32 + ND + + ND +++ ND B-29 +++ + +++ ND +++ +++ ND+++ ND B-30 +++ + >32 ND +++ +++ ND +++ ND B-31 ++ >32 +++ ND ND ND ND+++ ND B-32 + >32 ++ ND ++ + ND ++ ND B-33 + >32 ++ ND ++ + ND +++ NDB-34 +++ >32 +++ ND +++ +++ ND +++ ND B-35 +++ >32 +++ ND +++ +++ ND +++ND B-36 >32 >32 >32 + >32 >32 ND + >32 B-37 +++ >32 +++ ND +++ >32 ND+++ ND B-38 + >32 + ND ND ND ND + ND B-39 +++ >32 +++ ND +++ ++ ND +++ND B-40 ++ >32 +++ ND ++ ++ ND +++ ND B-41 +++ >32 +++ +++ +++ +++ ++++++ +++ B-42 +++ >32 +++ ND +++ +++ ND +++ ND B-43 + >32 + ND + + ND +ND B-44 +++ >32 ++ ND +++ ++ ND +++ ND B-45 ++ >32 ++ ND ND ND ND +++ NDB-46 +++ >32 +++ ND ND ND ND +++ ND B-47 +++ >32 +++ +++ ND +++ +++ ++++++ B-48 +++ >32 +++ +++ +++ ++ +++ +++ +++ B-49 +++ >32 +++ +++ +++ ++++++ +++ +++ B-50 +++ +++ +++ ND +++ +++ ND +++ ND B-51 ++ >32 >32 ND++ + ND + ND B-52 >32 >32 >32 ND ND ND ND >32 ND B-53 +++ >32 +++ ++++++ +++ +++ +++ +++ B-54 ++ >32 ++ ND +++ ++ ND +++ ND B-55 +++ >32 +++ND ND ND ND +++ ND B-56 +++ >32 +++ ND +++ +++ ND +++ ND B-57 + >32 +++ND ND ND ND +++ ND B-58 +++ >32 +++ + +++ +++ ++ +++ +++ B-59 + >32 ++ND ND ND ND ++ ND B-60 + >32 + ND ND ND ND + ND B-61 ND >32 >32 ND >32ND ND ND ND B-62 + >32 ++ ND +++ +++ ND ++ ND B-63 + >32 >32 ND +++ +++ND +++ ND B-64 +++ +++ +++ +++ +++ +++ +++ +++ +++ B-65 ++ >32 + ND + ++ND ++ ND B-66 + >32 >32 ND >32 >32 ND >32 ND B-67 +++ >32 +++ ND ND NDND +++ ND B-68 +++ >32 +++ +++ +++ +++ +++ +++ +++ B-69 + >32 + ND + >32ND +++ ND B-70 + >32 ++ ND ND ND ND + ND B-71 + >32 ++ ND ND ND ND +++ND B-73 +++ >32 >32 ND ++ ++ ND ND ND B-79 >32 >32 >32 ND >32 >32 ND NDND B-80 +++ >32 +++ ND +++ +++ ND ND ND B-81 >32 >32 >32 ND >32 >32 NDND ND B-82 +++ >32 +++ ND +++ +++ ND +++ ND B-83 +++ >32 +++ ND +++ +++ND +++ ND B-84 +++ + +++ ND +++ +++ ND +++ ND B-85 +++ + +++ ND +++ +++ND +++ ND B-86 +++ >32 +++ ND +++ +++ ND +++ ND C-1 + >32 ++ ND ++ + ND+++ ND C-2 ++ >32 ++ ND ++ ++ ND +++ ND C-3 + >32 ++ ND + + ND +++ NDC-4 + >32 + ND + + ND +++ ND C-5 + >32 + ND + + ND +++ ND C-6 + >32 +ND + + ND +++ ND C-7 + >32 + ND ++ + ND +++ ND C-8 +++ +++ +++ ND ++++++ ND +++ ND C-9 +++ +++ +++ +++ +++ +++ +++ +++ +++ C-10 +++ + +++ ND+++ +++ ND +++ ND C-11 +++ >32 ++ ND ++ +++ ND +++ ND C-12 +++ >32 +++ND +++ +++ ND +++ ND C-13 +++ >32 +++ ND +++ +++ ND +++ ND C-14 +++ >32++ ND +++ +++ ND +++ ND C-15 ++ >32 + ND ++ +++ ND +++ ND C-16 +++ >32+++ ND +++ +++ ND +++ ND C-17 +++ >32 +++ ND +++ +++ ND +++ ND C-18+++ >32 +++ ND +++ +++ ND +++ ND C-19 +++ >32 +++ ND +++ +++ ND +++ NDC-20 >32 >32 >32 ND >32 >32 ND +++ ND C-21 ++ >32 ++ ND ++ +++ ND +++ NDC-22 +++ + +++ ND +++ +++ ND +++ ND C-23 +++ >32 ++ ND +++ +++ ND +++ NDC-24 ++ >32 ++ ND ++ +++ ND +++ ND C-25 >32 >32 >32 ND >32 >32 ND +++ NDD-1 +++ >32 +++ ND +++ +++ ND +++ ND D-2 +++ + +++ +++ + +++ +++ +++ +++D-3 +++ +++ +++ + +++ +++ +++ +++ +++ D-4 >32 >32 >32 ND >32 >32 ND >32+++ D-5 +++ >32 +++ ND +++ +++ ND +++ ND D-6 + >32 + ND >32 + ND +++ NDD-7 +++ + +++ ND +++ +++ ND +++ ND D-8 +++ >32 +++ +++ +++ +++ +++ ++++++ D-9 +++ >32 +++ +++ +++ +++ +++ +++ +++ D-10 +++ ++ +++ +++ +++ ++++ +++ +++ D-11 +++ +++ +++ +++ +++ +++ +++ +++ +++ D-12 +++ +++ +++ +++++ +++ +++ +++ +++ D-13 +++ + +++ ++ +++ +++ +++ +++ +++ D-14 +++ >32+++ ND +++ +++ ND +++ ND D-15 +++ +++ +++ ND +++ +++ ND +++ ND D-16 ++++++ +++ ND +++ +++ ND +++ ND D-17 +++ +++ +++ ND +++ +++ ND +++ ND D-18+++ + +++ ND +++ +++ ND +++ ND D-19 +++ ++ +++ ND +++ +++ ND +++ ND D-20+++ ++ +++ ND +++ +++ ND +++ ND D-21 ++ ++ +++ ND +++ +++ ND +++ ND D-22+++ ++ +++ ND +++ +++ ND +++ ND D-23 +++ ++ +++ ND +++ +++ ND +++ NDD-24 +++ +++ +++ ND +++ +++ ND +++ ND D-25 +++ + +++ ND +++ +++ ND +++ND D-26 +++ + +++ ND +++ +++ ND +++ ND D-27 ++ >32 +++ ND + + ND +++ NDD-28 + >32 + ND + + ND +++ ND D-29 ++ >32 ++ ND + + ND +++ NDE-1 + >32 + ND ++ ++ ND +++ ND E-2 +++ >32 +++ +++ +++ +++ +++ +++ +++E-3 + >32 + ND >32 + ND + ND E-4 >32 >32 >32 ND + + ND ++ NDE-5 >32 >32 >32 ND + + ND >32 ND E-6 + >32 >32 ND ++ ++ ND ++ ND E-7+++ >32 +++ +++ +++ +++ +++ +++ +++ E-8 + >32 +++ ND +++ +++ ND +++ NDE-9 >32 >32 + ND >32 >32 ND + ND E-10 +++ >32 +++ ND +++ +++ ND +++ NDE-11 +++ >32 +++ ND +++ +++ ND +++ ND E-12 ++ >32 ++ ND ++ ++ ND +++ NDE-13 >32 + >32 ND + ++ ND +++ ND E-14 +++ ++ +++ ND +++ +++ ND +++ NDE-15 ++ ++ ++ ND +++ +++ ND +++ ND E-16 +++ +++ >32 ND +++ +++ ND +++ NDE-17 +++ >32 >32 ND +++ +++ ND +++ ND E-18 +++ >32 +++ ND +++ +++ ND +++ND E-19 +++ >32 +++ ND +++ +++ ND +++ ND F-1 +++ >32 +++ ND +++ +++ ND+++ ND F-2 + ND + ND + >32 ND >32 ND F-3 + >32 ++ ND + + ND +++ NDF-5 >32 >32 >32 ND >32 >32 ND +++ >32 F-6 >32 >32 + ND >32 >32 ND +++ NDF-7 >32 >32 + ND + + ND +++ ND F-8 ++ + + ND ++ ++ ND +++ NDF-9 >32 >32 >32 ND + + ND + ND F-10 ++ + ++ ND ++ ++ ND + ND G-1 +++ ++++++ ND +++ +++ ND +++ ND G-2 +++ +++ +++ ND +++ +++ ND +++ ND G-3 ++ + +ND + ++ ND ++ ND

In Vivo Biological Data

This example demonstrates in vivo efficacy against infection bymethicillin resistant Staphylococcus aureus ATCC 33591, using a murineneutropenic thigh model.

A S. aureus ATCC 33591 culture was grown to log phase overnight anddiluted in phosphate buffered saline (pH 7.2) to an optical density ofabout 0.1 at 600 nm, giving an approximate concentration of 10⁸ cfu/mL.The suspension was diluted 1:100 in phosphate buffered saline (pH 7.2)for a final concentration of 10⁶ cfu/mL.

Outbred female CF1 mice (approx. 20 gram body weight) were renderedneutropenic by treatment with cyclophosphamide (200 mg/kg body weight,intraperitoneal injection) at 2 and 4 days prior to inoculation. Groupsof 5 mice were inoculated with 0.05 mL of the bacteria (approx. 10⁶cfu/mL) into the anterior thigh. Each group was treated intravenouslytwo hours post infection with vehicle (phosphate buffered saline) ortest compound. The mice were sacrificed at either 6 or 24 hrs aftertreatment and thighs were collected aseptically. Each thigh was weighed,placed into sterile saline, and homogenized. The tissue homogenates werediluted appropriately for plating on agar plates. Colony counts wererecorded (cfu/gram) and compared to control groups. The data arepresented in Table F below:

TABLE F Murine Neutropenic Thigh Model Colony Count Compound No. Dose(log cfu/gram) (Time) (mg/kg) Compound Vehicle A-4 (6 hr) 50 4.99 8.02A-4 (6 hr) 25 5.75 7.99 A-6 (6 hr) 50 6.65 8.02 A-8 (6 hr) 50 4.45 8.12A-8 (6 hr) 25 5.85 8.12 A-9 (6 hr) 50 5.11 8.02 A-9 (6 hr) 25 7.11 8.67A-26 (6 hr) 50 5.34 7.74 A-26 (6 hr) 25 6.37 8.04 B-6 (6 hr) 50 6.378.17 B-47 (6 hr) 50 7.53 8.54 B-50 (6 hr) 50 6.58 8.76 B-56 (6 hr) 506.38 7.65 B-67 (6 hr) 50 7.73 8.76 C-12 (6 hr) 50 5.93 7.88 D-3 (6 hr)50 6.28 7.99 D-11 (6 hr) 50 6.25 7.99 E-15 (6 hr) 50 5.77 7.95

In vivo efficacy was shown by a decrease in colony count (log cfu/gramof tissue) in the compound-treated animals when compared against thecolony count in animals given only the vehicle.

DNA Binding

This example illustrates the DNA binding properties of compounds of thisinvention using a DNase I footprinting technique. Generally, theprocedure described in Dervan, WO 98/50582 (1998), was followed.

Double stranded circular plasmids A and B were used to prepare doublestranded DNA-binding probes containing the target sequences for theDNase I footprint titration experiments.

Plasmid A was prepared by hybridizing two sets of 5′-phosphorylatedcomplementary oligonucleotides, the first set being

5′-CTAGATGCCGCTAAGTACTATGCCGCTAACTACTATGCCGCTAAT TACTATGCCGC-3′and

5′-CATAGTAATTAGCGGCATAGTAGTTAGCGGCATAGTACTTAGCG GCAT-3′;and the second set being

5′-TAAATACTATGCCGCTAACTAGTATGCCGCTATGCA-3′and

5′-TAGCGGCATACTAGTTAGCGGCATAGTATTTAGCGG-3′,and ligating the resulting duplexes to the large pUC19 XbaI/PstIrestriction fragment.

Plasmid B was the plasmid pTrc99a, obtained from Amersham PharmaciaBiotech, Inc.

The 3′-³²P end-labeled EcoRI/PvuII fragments from each plasmid wereprepared by digesting the plasmids with EcoRI and PvuII withsimultaneous fill-in using Sequenase v. 2.0,[α-³²P]-deoxyadenosine-5′-triphosphate, and[α-³²P]-thymidine-5′-triphosphate, and isolating the cloned fragments bynondenaturing gel electrophoresis. A and G sequencing reactions werecarried out as described (See Maxam and Gilbert, Methods Enzymol., 1980,65, 499-560; Iverson and Dervan, Methods Enzymol., 1987, 15, 7823-7830;Sambrook et al., 1989, Molecular Cloning, 2^(nd) ed., Cold Spring HarborLaboratory Press: Cold Spring Harbor, N.Y.) Standard methods were usedfor all DNA manipulations (Sambrook et al., ibid.)

The 310 base pair dsDNA restriction fragment (SEQ ID NO. I) of Plasmid Acontained a target sequence AGTACT. The 352 base pair dsDNA restrictionfragment (SEQ ID NO. II) of Plasmid B contained target sequencesACAATTAT and AATTAATCAT. These fragments were used for quantitativeDNase I footprinting experiments. The target sequences were selected fortheir identity with, or similarity to, promoter sites for bacterialgenes.

Quantitative DNase I footprint titration experiments were carried out asdescribed previously (Dervan, WO 98/50582, 1998) with the followingchanges. All reactions were carried out in a total volume of 400 μL,with compound stock solution or water added to 15,000 cpm radiolabeledrestriction fragment affording final solution conditions of 10 mMTrisHCl, 10 mM KCl, 10 mM MgCl₂, 5 mM CaCl₂, pH 7.0 and 0.01 nM, 0.1 nM,1.0 nM, 10.0 nM compound or no compound for reference lanes. Thecompounds were allowed to equilibrate at 22° C. for 16 hr. Footprintingreactions were initiated with addition of 10 μL of a DNase I stocksolution (at the appropriate concentration to give ˜50% intact DNA)containing 1 mM DTT and allowed to proceed for 7 min at 22° C. Thereactions were stopped, ethanol precipitated, resuspended in loadingbuffer, heat denatured, and placed on ice as described previously(Dervan WO 98/50582, 1998). The reaction products were separated on aprecast 8% polyacrylamide denaturing sequencing Castaway gel with 32preformed wells from Stratagene in 1×TBE at 2000 V. Gels were driedaccording to the manufacturer and exposed to a storage phosphor screen(Molecular Dynamics). Quantitation and data analysis were carried out asdescribed in Dervan, WO 98/50582, 1998.

dsDNA binding results are provided in Table G:

TABLE G dsDNA Binding Dissociation Com- Target Constant Target Locationpound Sequence K_(d) (nM) (Fragment/Plasmid). A-3 AATACT 5 310 bp/A A-3AATTAATCAT 1 352 bp/B B-18 AGTACT 50 310 bp/A B-18 AATTAATCAT 2 352 bp/B

SEQUENCE LISTINGS SEQ ID NO. I (310 by EdoRI/PvuII restrictionfragment from Plasmid A; only one strand shown)AATTCGAGCTCGGTACCCGGGGATCCTCTAGATGCCGCTAAGTACTATGCCGCTAACTACTATGCCGCTAATTACTATGCCGCTAAATACTATGCCGCTAACTAGTATGCCGCTATGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGSEQ ID NO. II (352 by EdoRI/PvuII restrictionfragment from Plasmid B; only one strand shown)CTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCGCGAATTGATCTGGTTTGACAGCTTATCATCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCA CACAGGAAACAGACCATGGAATT

Synthesis of Compounds General

Typically, compound structures were confirmed by ¹H-NMR and/or massspectrometry. Where a parenthetical remark such as “¹H-NMR” or “massspectrum” follows without elaboration a reference to a compound, itmeans that such spectrum was taken, was consistent with the assignedstructure, and did not indicate the presence of significant impurities.

Abbreviations or acronyms in common usage are employed for varioussolvents, catalysts and reagents, including: TFA for trifluoroaceticacid, NMP for N-methylpyrrolidone, DMF for N,N-dimethylformamide, MsClfor methanesulfonyl chloride, triflate for trifluoromethanesulfonate,HBTU for 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate; DIEA for diisopropylethylamine; HOBt forhydroxybenzotriazole; DCC for dicyclohexylcarbodiimide; BOPCl forbis(2-oxo-3-oxazolidinyl)-phosphinic chloride; HATU forO—(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate; Boc₂O for Boc anhydride; and MOMCl formethoxymethyl chloride.

The skilled artisan will understand that: (a) an intermediate orsynthetic route described in the context of the synthesis of aparticular compound of this invention can also be used to make othercompounds of this invention, mutatis mutandis; (b) in certainexperimental sections only the preparation of an intermediate compoundis described, because its incorporation into a compound of thisinvention straightforwardly follows synthetic methodology describedelsewhere herein; and (c) for some routine reactions that recur herein,a detailed description of each set of reaction and work-up conditionsmay be replaced by a reference to a general procedure in the interest ofbrevity, the conditions described in the general procedure beingadaptable to the instance at hand without undue experimentation.

General Procedures

Procedure A: activation of a heterocyclic carboxylic acid with HBTU (orHATU) and coupling to a heterocyclic amine. The acid (1.2 equiv.) andHBTU (or HATU, for less reactive amines) (1.14 equiv.) were dissolved inNMP (or DMF, dilution: ca. 1 g/10 mL) and DIEA (ca. 1/10 of the volumeof NMP/DMF), stirred for 30-60 min at 25-40° C. and added to a solutionof the amine (1 equiv.) in NMP (or DMF, dilution: ca. 1 g/5 mL) and DIEA(ca. 1/10 of the volume of NMP/DMF). The mixture was stirred for 12 to16 h at 25-40° C.

Procedure B: activation of a heterocyclic carboxylic acid with HBTU andcoupling to an aliphatic amine. A heterocyclic acid (1 equiv.) and HBTU(1.1 to 1.2 equiv.) were dissolved in NMP (or DMF, dilution: ca. 1 g/10mL) and DIEA (ca. 1/10 of the volume of NMP/DMF), stirred for 30-60 minat 25-40° C. and treated with the aliphatic amine (excess, ca. 2 to 50equiv.). The mixture was stirred for 12 to 16 h at 25-40° C.

Procedure C: activation of a heterocyclic carboxylic acid with BOPCl andcoupling to an aliphatic amine. A heterocyclic acid (1 equiv.) and BOPCl(1.2 equiv.) were dissolved in NMP (or DMF, dilution: ca. 1 g/10 mL) andDIEA (ca. 1/10 of the volume of NMP/DMF), stirred for 30-60 min at25-40° C. and treated with the aliphatic amine (excess, ca. 2 to 50equiv.). The mixture was stirred for 12 to 16 h at 25-40° C.

Procedure D: coupling of a heterocyclic acyl chloride to a heterocyclicamine. A mixture of the heterocyclic acid chloride (1.0 to 1.2 equiv.)and the heterocyclic amine in NMP (or DMF, dilution: ca. 1 g/10 mL) andDIEA (ca. 1/10 of the volume of NMP/DMF) was stirred at 25-60° C. for 12to 16 h.

Procedure E: workup and purification by HPLC. The crude product wasdiluted with an approximately 3× volume of a mixture of AcOH/H₂O (1:1)and purified by preparative HPLC. The product was characterized by¹H-NMR (d₆-DMSO) and ESI-MS.

Procedure F: workup and purification by HPLC. The crude product wasdiluted with an approximately 7× volume of a mixture of AcOH/H₂O (1:1)and washed with Et₂O (1 to 3 times). The aqueous layer was purified bypreparative HPLC. The product was characterized by ¹H-NMR (d₆-DMSO) andESI-MS.

Procedure G: precipitation and filtration. The reaction mixture wasadded dropwise to ice-water (ca. 10 to 15 times the volume of thereaction mixture). The resulting precipitate was collected by filtrationand dried to give the crude product as a solid.

Procedure H: saponification of a heterocyclic carboxylic acid ester. Amixture of the ester and NaOH or KOH (ca. 0.5 g base per g ester) in H₂O(ca. 1 g ester per 40 mL) and MeOH or EtOH (1 g ester per 20 mL) wasstirred at 40-70° C. for 3 to 12 h. The reaction mixture was dilutedwith H₂O (ca. twice the volume of the reaction mixture), washed withAcOEt (1×) and acidified to pH=2-3 using ca. 6M aqueous HCl. Theresulting precipitate was collected by filtration and dried.

Procedure I: hydrogenation of an aryl nitro group to an aryl amine andisolation of the amine as the hydrochloride. A suspension of the nitrocompound and 10% Pd—C (5-10 wt % with respect to the nitro compound) ina mixture of MeOH/AcOEt (ca. 1:10 to 1:1) was stirred at roomtemperature (RT) under H₂ atmosphere (1 atm to 150 psi) for 10-20 hr andfiltered through Celite. The filtrate was concentrated in vacuo, dilutedwith Et₂O, and treated with HCl (g). The resulting precipitate wascollected by filtration and dried, or in case no precipitate was formed,the crude reaction mixture was evaporated to dryness.

Example A

This example relates to the synthesis of compounds according to formula(Ic), such as compounds A-3 to A-16, A-24, A-26, A-29 (Scheme 1) andA-17 to A-23, A-25 (Scheme 2). Schemes 1 and 2 illustrate the synthesisof compounds A-3 and A-17, respectively.

Trimer 3. A solution of ketone 1 (60.0 g, 1.0 equiv.) and amine 2 (72.7g, 1.0 equiv.) in NMP (500 mL) and DIEA (95 mL) was stirred for 2 h atRT (slight exotherm). The reaction mixture was added dropwise toice-water (1.5 L), the resulting precipitate collected by filtration anddried to give trimer 3 (89 g, 92%, dark powder).

Amine 4. Trimer 3 (11.0 g, 1 equiv.) was hydrogenated using Procedure Ito give amine 4 (8.75 g, 78%).

Tetramer 6. Carboxylic acid 5a (1.45 g, 1.2 equiv.) and amine 4 (2.9g, 1. equiv.) were coupled using Procedure A to give tetramer 6 as adark powder (2.77 g, 74%, ¹H-NMR). (4-Chloro-2-fluoro-benzoyl chloride(5b) can be used instead of acid (5a)/HBTU.)

Acid 7. Tetramer 6 (2.77 g, 1 equiv.) was saponified using Procedure Hto give acid 7 (2.06 g, 76%).

Compound A-3. Acid 7 (0.62 g, 1 equiv.) and 4-(2-aminoethyl)-morpholine(5 mL) were coupled according to Procedure B using HBTU (490 mg, 1.1equiv.). Purification according to Procedure F gave compound A-3 (290mg).

Example B

Scheme 2 describes the preparation of additional compounds (Ic), wherethe subscript i is 2. Intermediates 9 to 12 were prepared similarly tointermediates 3, 4, 6 and 7, respectively (Scheme 1). Similarly to thesynthesis of compound A-3, compounds such as A-17 to A-23 and A-25 wereprepared using Procedures B and F or Procedures C and E.

Example C

Scheme 3 illustrates an alternative synthesis of compounds (Ic). Thefinal step is the coupling of trimeric amine 15 with various benzoicacids (e.g., 16 to 19) according to the Procedures A and F to yield thefinal products A-1, A-30, A-34 and A-35, respectively.

Example D

The synthesis of compounds bearing a quaternized methyl morpholiniummoiety at the C-terminus is illustrated in Scheme 4 with specificreference to compound A-28. Compound B-30 was prepared analogously.

Compound 21. A solution of morpholino compound 20 (1.00 g, 1.0 equiv.)in CH₂Cl₂ (10 mL) was treated at RT with methyl triflate (0.337 mL, 1.05equiv.) and stirred for 2 h. The mixture was treated with DIEA (0.5 mL)and additional methyl triflate (0.3 mL). Stirring was continued for 1½ hand the solvent was evaporated. The ¹H-NMR spectrum of the crude productwas in agreement with the structure of compound 21, but showed minorimpurities. The material was used without further purification.

Compound 22. A solution of crude compound 21 in MeOH (ca. 40 mL) wassaturated with HCl (g) for about 20 seconds and stirred at RT for 1 h.Evaporation of the solvent gave compound 22 (¹H-NMR, mass spectrum).

Compound A-28. Trimeric acid 12 (79.9 mg, 1.2 equiv.) and compound 22(60 mg, 1.0 equiv.) were coupled according to Procedure A using HBTU(76.3 mg, 1.14 equiv.) and purified according to Procedure F to givecompound A-28.

Example E

Scheme 5 shows an efficient synthetic approach for compounds (Id), withspecific reference to compound B-11. The scheme is applicable to othercompounds such as B-3 to B-22, B-24 to B-29, B-31 to B-34 to B-38 toB-50, B-52 to B-62, B-65, and B-67 to B-71.

Ester 31. 2,4-Difluorobenzoyl chloride (2.16 mL, 1.1 equiv.) and aminoester 2 (5.00 g, 1.0 equiv.) were coupled according to Procedure D.Workup according to Procedure G gave ester 31 as a white solid (4.94 g,74%, ¹H-NMR).

Acid 32. Ester 31 (4.94 g, 1.0 equiv.) was saponified according toProcedure H to give acid 32 (4.44 g, 92%, ¹H-NMR) as a white solid.

Compound B-11. Acid 32 (100 mg, 1.05 equiv.) and4(2-aminoethyl)-thiomorpholine (300 μl) were coupled according toProcedure B using HBTU (90 mg, 1.0 equiv.). Purification according toProcedure F gave compound B-11.

Example F

Compounds bearing a guanidinium group at the C-terminus were preparedfrom the corresponding primary amine according to Scheme 6 (exemplifiedwith compound B-23).

Compound B-23. A solution of the primary amine 35 (10 mg, ca. 1 equiv.)and pyrazole 40 (ca. 4.5 mg, ca. 6 equiv.) in DMF (0.3 mL) and DIEA(0.03 mL) was stirred at RT for 12 h. Workup according to Procedure Eyielded compound B-23.

Example G

Scheme 8 describes another synthetic approach to compounds according toformula (Id) (i=3). Either an in situ activated benzoic ester (OBtester) or an acid chloride can be used as a coupling partner for amine54. Compounds B-72 to B-80 were analogously synthesized.

Compound B-47. Benzoic acid 5 (46 mg, 1.2 equiv.) and amine 54 (110 mg,1.0 equiv.) were coupled according to Procedure A using HBTU (96 mg,1.14 equiv.). Purification according to Procedure F gave compound B-47.

Example H

Scheme 9 illustrates the synthesis of compounds (Id) (i=2), using anapproach analogous to that of Scheme 8: either a benzoic acid (e.g. 5a)was coupled to the amine 13 according to Procedure A and worked upaccording to Procedure F or a benzoyl chloride was coupled to 13according to Procedure D and worked up according to Procedure E.

Example I

Scheme 11 depicts the synthesis of carboxylic acid intermediates thatcan be coupled with an amine to make compounds of this invention, asdescribed later herein.

Specific procedures are given below for carboxylic acid 82, but acids12, 32, and 83-87 were prepared analogously.

Ester 81. 4-Chloro-2-fluorobenzoic acid 16 (3.77 g, 1.2 equiv.) andamino ester 80 (3.42 g, 1.0 equiv.) were coupled according to ProcedureA using HBTU (7.77 g, 1.14 equiv.). Workup according to Procedure Gyielded ester 81, which was used for the next step without furtherpurification.

Compound 82. Crude ester 81 was saponified according to Procedure H toyield carboxylic acid 82 (4.72 g, 88% over two steps, ¹H-NMR).

Example J

Scheme 12 describes the synthesis of carboxylic acid building blockscontaining a pyrrole unit that bears a 3-hydroxypropyl, an ethyl or acyclopropylmethyl group at the ring nitrogen. Experimental details aregiven for the synthesis of compounds 101, 102, 113 and 114, otherbuilding blocks being prepared analogously.

Compound 101. A suspension of pyrrole 100 (20.0 g, 1.0 equiv.),3-bromopropanol (13.75 mL, 1.4 equiv.), NaI (16.28 g, 1.0 equiv.) andK₂CO₃ (30.0 g, 2.0 equiv.) in DMF (200 mL) was stirred at 65° C. for 2h. The mixture was poured into H₂O (700 mL) and extracted with Et₂O(3×). The combined organic layers were dried (MgSO₄) and evaporated togive compound 101 (27.2 g, ¹H-NMR).

Compound 102. Compound 101 (18 g) was hydrogenated according toProcedure I to give compound 102 (16.6 g, 92%, ¹H-NMR).

Compound 113. 2,4-Difluorobenzoyl chloride (3.12 g, 1.1 equiv.) andamine 102 (4.00 g, 1.0 equiv.) were coupled according to Procedure D.The mixture was treated with H₂O (300 mL) and sat. aqueous K₂CO₃ (25 mL)and extracted with AcOEt (2×). The combined organic layers were dried(MgSO₄) and evaporated to give compound 113 as an orange oil that wasused without further purification.

Compound 114. Crude compound 113 was saponified according to Procedure Hto give compound 114 as pale yellow solid (¹H-NMR).

Example K

Scheme 13 describes the synthesis of the heterocyclic amines containingan unsubstituted pyrrole carboxamide unit. These amines can be coupledwith carboxylic acids to make compounds of this invention, as describedlater herein.

Compounds 122/123. Carboxylic acid 120 (5.00 g, 1.0 equiv.) and4-(2-aminoethyl)morpholine (4.35 mL, 1.5 equiv.) were coupled accordingto Procedure B using HBTU (9.22 g, 1.1 equiv.). The reaction mixture waspoured into ice-water (ca. 300 mL) and sat. aqueous K₂CO₃ (20 mL) andextracted with Et₂O (4×). The combined organic layers were dried (MgSO₄)and evaporated. Flash chromatography (1% Et₃N in CH₂Cl₂/0 to 10% MeOH)gave compound 122 (760 mg). A solution of compound 122 (760 mg) in AcOEt(20 mL) was treated with HCl-saturated AcOEt (75 mL) and stirred for 3 hat 0° C. The solids were collected by filtration and dried to givecompound 123 (615 mg, ¹H-NMR).

Example L

Scheme 14 depicts the synthesis of heterocyclic amine building blockscontaining a hydroxypropyl substituent at a pyrrole nitrogen.

Compound 130. A solution of amine 102 (31.75 g, 1.0 equiv.) and Boc₂O(26.87 g, 1.2 equiv.) in dioxane (100 mL) was treated with a sat. aq.Na₂CO₃ (12 g), stirred at RT for 24 h, diluted with H₂O, and extractedwith AcOEt (4×). The combined organic layers were dried (MgSO₄) andevaporated to give compound 130 as a red tar (¹H-NMR).

Acid 131. Compound 130 (ca. 34 g) was saponified using Procedure H togive acid 131 as a sticky solid (¹H-NMR).

Compounds 133/134. Acid 131 (1.54 g, 1.1 equiv.) and amine 132 (1.60 g,1.0 equiv.) were coupled according to Procedure A using HBTU (1.96 g,1.05 equiv.). The reaction mixture was poured into H₂O (150 mL) and sat.aqueous K₂CO₃ (20 mL) and extracted with AcOEt (4×). The organic layerswere dried (MgSO₄) and evaporated to give compound 133 as an orange tarthat was used without further purification. A solution of the crudecompound 133 in AcOEt (ca. 60 mL) was treated with HCl (g), stirred for2.5 h at RT and evaporated to give amine 134 (1.06 g) as a hygroscopicbrown solid.

Compound 135. Amine 135 was prepared by coupling (Procedure A) acid 131and 1-(2-aminoethyl)piperidine followed by cleavage of the Boc-groupusing HCl.

Example M

Scheme 15 illustrates the synthesis of more intermediate amines having apyrrole bearing a substituent other than methyl on the ring nitrogen,with compound 142 as an example.

Nitro compound 141. A mixture of dimer 140 (1.00 g, 1.0 equiv.), K₂CO₃(885 mg, 2.5 equiv.) and 1-chloro-3-iodopropane (0.44 mL, 1.6 equiv.) inDMF (10 mL) was stirred at 65° C. for 3 h, poured into H₂O (135 mL) andsat. aqueous K₂CO₃ (20 mL) and extracted with AcOEt (3×). The organiclayers were dried (MgSO₄) and evaporated to give nitro compound 141(0.86 g, 72%, ¹H-NMR, mass spectrum).

Amine 142. Nitro compound 141 (0.86 g) was hydrogenated using ProcedureI to give amine 142 as a brown solid (0.923 g, >95%).

Example N

Scheme 16 describes an alternative synthesis of intermediate amineshaving a substituent on a pyrrole nitrogen.

Ester 151. A mixture of ethyl 4-nitropyrrole-2-carboxylate 150 (20.00 g,1.0 equiv.), 4-(2-chloroethyl)morpholine hydrochloride (28.28 g, 1.4equiv.), NaI (16.28 g, 1.0 equiv.) and K₂CO₃ (28.78 g, 1.92 equiv.) inDMF (200 mL) was stirred at 60° C. for 10.5 h and poured into a mixtureof H₂O and saturated aq. K₂CO₃ (550/50 mL). The resulting solution wasextracted with AcOEt (4×, each 200 mL). The combined organic layers weredried (MgSO₄) and evaporated to give ester 151 as pale yellow crystals(31.4 g, 97%, ¹H-NMR).

Acid 152. Ester 151 (31.4 g, 1.0 equiv.) was saponified using ProcedureH to give acid 152 as a white solid (23.0 g, 81%, ¹H-NMR).

Compounds 153/154. Acid 152 (1.04 g, 1.1 equiv.) and amine 132 (1.00 g,1.0 equiv.) were coupled according to Procedure A using HBTU (1.22 g,1.05 equiv.). The reaction mixture was poured into H₂O (150 mL) and sat.aqueous K₂CO₃ (20 mL) and extracted with AcOEt (5×). The combinedorganic layers were dried (MgSO₄) and evaporated to give compound 153,which was then hydrogenated using Procedure I to give compound 154,which was used without further purification.

Example O

Scheme 17 describes the synthesis of additional building blocks having asubstituent on a pyrrole ring nitrogen. Intermediates 160 to 163 weremade by analogy to intermediates 151 and 152 (vide supra). Compounds164, 166 and 168 were prepared by coupling 1-(2-aminoethyl)piperidine tothe corresponding acids 161, 152, and 162 using Procedure B.Intermediates 165, 167 and 169 were prepared by hydrogenation of thecorresponding nitro-pyrroles 164, 166, and 168, respectively.

Example P

Scheme 18 describes the synthesis of a building block having amethoxy-methyl substituent on a pyrrole ring nitrogen.

Compounds 181/182. Carboxylic acid 180 (1.66 g, 1.1 equiv.) and amine 13(2.50 g, 1.0 equiv.) were coupled according to Procedure A using HBTU(2.22 g, 1.05 equiv.). The crude product was poured into H₂O (300 mL)and extracted with AcOEt (4×). The combined organic layers were dried(MgSO₄) and evaporated to give a brown oil. Flash chromatography (CH₂Cl₂and 0→15% MeOH gradient) gave 181 (1.7 g) as a glassy solid. Thematerial was dissolved in AcOEt (50 mL), treated with HCl-saturatedAcOEt (50 mL) and stirred at RT for 1½ h. Evaporation of the solventgave amine 182 (1.49 g) as a tan solid. The ¹H-NMR spectrum and the massspectrum were consistent with the structure of amine 182. However, the¹H-NMR spectrum showed signals of minor impurities that were not furthercharacterized.

Example Q

Applying convergent synthesis principles to the amine and acid buildingblocks whose syntheses were described in preceding Examples I through Pabove and to building blocks 190 through 192 shown below, compounds ofthis invention were made as tabulated in Table H.

TABLE H Carboxylic Procedure Procedure acid Amine (coupling)(purification) Product 32 123 A F C-1 83 125 A F C-2 85 125 A F C-3 83133 A F C-4 85 133 A F C-5 114 13 A F C-6 190 182 D E C-7 12 123 A F C-812 134 A F C-9 82 125 A F C-10 86 125 A F C-11 110 191 A F C-12 112 191A F C-13 82 133 A F C-14 86 133 A F C-15 108 13 A F C-16 5b 182 D E C-1782 142 A F C-18 86 142 A F C-19 32 165 A F D-2 32 169 A F D-5 83 144 A FD-7 12 167 A F D-13 12 169 A F D-14 82 144 A F D-25 86 144 A F D-26 84155 A F D-27

Example R

This example illustrates the synthesis of compounds having a C-terminal3-methyl-isothiazole moiety. Also illustrated is the introduction of apendant charged side chain by mesylation of a pendant alcohol, followedby substitution using an amine.

Compound 201. A solution of ketone 1 (50.00 g, 1.05 equiv.) and amine200 (29.15 g, 1.0 equiv.) in DMF (300 mL) and pyridine (75 mL) wasstirred at 65° C. for 22 h. Workup according to Procedure G gave dimer201 (48.9 g, >95%, ¹H-NMR).

Compound 202. Dimer 201 (5.0 g) was hydrogenated using Procedure I togive compound 202 (¹H-NMR).

Ester 203 and acid 204. Acid chloride 5b (3.41 g, 1.1 equiv.) and aminoester 102 (4.00 g, 1.0 equiv.) were coupled according to Procedure D.The reaction mixture was diluted with H₂O (300 mL) and sat. aqueousK₂CO₃ (25 mL), extracted with AcOEt (2×), dried (MgSO₄) and evaporatedto give ester 203 as an orange oil. Saponification of crude ester 203according to Procedure H gave acid 204 as a white solid (¹H-NMR).

Alcohol 205. Acid 204 (722 mg, 1.1 equiv.) and amine 202 (0.50 g, 1.0equiv.) were coupled according to Procedure A using HBTU (0.77 g, 1.05equiv.). The reaction mixture was poured into H₂O (150 mL) and extractedwith AcOEt (7×). The combined organic layers were dried (MgSO₄) andevaporated. Flash chromatography (CH₂Cl₂/0%→15% MeOH gradient) gavealcohol 205 (358 mg, 34%, ¹H-NMR, mass spectrum).

Compounds 206/D-16. A solution of alcohol 205 (25 mg, 1.0 equiv.) in DMF(1 mL) and DIEA (0.1 mL) was treated at RT with MsCl (15.8 mg, 3equiv.), stirred for 70 min, treated with pyrrolidine (0.5 mL) andstirred at 55° C. for 16 h to give compound D-16 after purificationaccording to procedure E.

Example S

Schemes 21 and 22 describe the synthesis of C-terminal building blockscontaining a 3-methyl-isothiazole unit.

Compounds 210/211. A solution of carboxylic acid 163 (1.57 g, 1.0equiv.) and HATU (2.21 g, 1.0 equiv.) in DMF (8 mL) and DIEA (1.5 mL)was stirred at RT for 30 min and added to a solution of isothiazole 200(0.88 g, 1.0 equiv.) in DMF (5 mL) and DIEA (1 mL). The mixture wasstirred at 60° C. for 8 h, treated with H₂O and extracted (AcOEt, 3×).The combined organic layers were dried (MgSO₄) and evaporated to givecrude compound 210, which was hydrogenated using Procedure H to givecompound 211, used without further purification.

Compounds 220/221. A suspension of dimer 201 (3.0 g, 1.0 equiv.),1-(2-chloroethyl)pyrrolidine (HCl, 2.83 g, 1.4 equiv.), NaI (1.96 g, 1.1equiv.) and K₂CO₃ (3.29 g, 2.0 equiv.) in DMF (30 mL) was stirred at 65°C. for 7 h, treated with additional 1-(2-chloroethyl)pyrrolidine (.HCl,3.0 g) and stirred for 30 h at 65° C. The reaction mixture was dilutedwith H₂O (150 mL) and sat. aqueous K₂CO₃ (10 mL) and extracted withAcOEt (5×). The combined organic layers were dried (MgSO₄) andevaporated to give crude product 220 (3.36 g). The ¹H-NMR indicated thatproduct 220 was a mixture of two compounds. The crude product 220 (3.36g) was hydrogenated using Procedure I to give compound 221 plus at leastone unidentified by-product, which was used without furtherpurification. Compounds 222 to 225 were prepared in a similar fashion.

The building blocks described above were coupled under standard couplingconditions as summarized in the following Table I.

TABLE I Carboxylic Procedure Procedure acid Amine (coupling)(purification) Product 83 211 A F D-1 85 211 A F D-4 82 211 A F D-8 86211 A F D-9 84 211 A F D-28 192 211 A F D-29 83 223 A F D-3 83 225 A FD-6 86 221 A F D-10 86 223 A F D-11 82 223 A F D-12 82 225 A F D-22 86225 A F D-23Compound G-2 was obtained as a by-product of the synthesis of compoundD-10, indicating that the unidentified by-product in the synthesis ofcompound 221 was a doubly alkylated bis-ethylpyrrolidine.

Example T

This example illustrates the synthesis of compounds having a C-terminal3-methyl-isothiazole moiety and an N-terminal benzamide unit thatcontains a halogen (F, Cl) at position 4 and a NHR or a SR substituentat position 2, using compound C-24 as a model. Generally, the compoundswere prepared from precursors having an N-terminal 2,4-difluorobenzamideor a 4-chloro-2-fluorobenzamide unit. In the presence of an appropriatenucleophile (e.g., amine, thiolate), these N-terminal units undergo anucleophilic aromatic substitution replacing the fluoride at position 2of the benzene ring.

Compound C-24. A mixture of tetramer 230 (prepared from theintermediates 82 and 202, respectively: 100 mg, 1.0 equiv.) and4-(2-aminoethyl)thiomorpholine (0.3 mL) in NMP (0.7 mL) was stirred at55° C. for 17 h. Purification according to Procedure E gave compoundC-24.

Example U

This example illustrates the synthesis of a building block having a2,4-disubstituted thiophene ring.

Compounds 241/242. A solution of ethyl-2-thiophene carboxylate 240 (200g, 1 mol) in TFA (200 mL) was slowly added to a mixture of TFA (900 mL)and fuming nitric acid (200 mL) at 5° C. The cooling was removed and thereaction mixture stirred at 45° C. for ca. 14 h, cooled to ca. 10° C.,and poured into vigorously stirred ice water (4 L). The resultingprecipitate was collected by filtration and washed with ice water (2×).Lyophilization gave a mixture of compounds 241/242 (2:3 by ¹H-NMR, 194.2g, 79%)

Compounds 243/244. The mixture of compounds 241/242 (2:3, 20 g) washydrogenated using Procedure I to give a mixture of compounds 243/244(0.9:1 as evidenced by ¹H-NMR, 19.02 g, 93%).

Acid 245. Compound 243 was selectively saponified by treating a mixtureof compounds 243/244 (0.9/1, 15 g) in methanol (500 mL) at 0° C. with asolution of KOH (9 g) in H₂O (75 mL) and stirring for 3 h. The reactionmixture was then diluted with H₂O (400 mL) and washed with EtOAc (3×,200 mL). The aqueous layer was neutralized to pH=6.5 with 1 M aq. HCl,treated with Na₂CO₃ (15 g) and a solution of Boc anhydride (15 g) indioxane (150 mL) and stirred for 24 h at RT. The reaction mixture waswashed with EtOAc (3×, 200 mL), cooled to 0° C., acidified to pH=2.8with aqueous HCl (50%), and extracted with EtOAc (3×, 200 mL). Thecombined organic layers were dried (MgSO₄) and evaporated. The remainingoil was dissolved in methanol and treated with activated carbon (5 g).The mixture was filtered through Celite and the filtrate evaporated toyield acid 245.

Example V

Scheme 25 illustrates the synthesis of compounds incorporating athiophene moiety from building block acid 245, compound E-14 as anexemplar.

Compounds 250/251. Acid 245 (4.93 g, 1.2 equiv.) and amino ester 8 (3.22g, 1.0 equiv.) were coupled according to Procedure A using HBTU (7.29 g,1.14 equiv.). Workup according to Procedure G gave compound 250, whichwas then dissolved in AcOEt (200 mL) at 0° C., treated with HCl (g) andstirred for 2 h at RT. The solids were collected by filtration to giveamino ester 251 (4.92 g, 92% two steps, ¹H-NMR).

Compounds 252/253. Acid 82 (1.07 g, 1.2 equiv.) and amino ester 251(1.00 g, 1.0 equiv.) were coupled according to Procedure A using HBTU(1.36 g, 1.14 equiv.). Workup according to Procedure G gave tetramer 252(1.86 g). According to the ¹H-NMR spectrum, this material containedminor impurities. Tetramer 252 (1.86 g, 1.0 equiv.) was saponified usingProcedure H to give acid 253 (0.75 g).

Compound E-14. Acid 253 (80 mg, 1.0 equiv.) and1-(2-aminoethyl)piperidine (0.5 mL) were coupled according to ProcedureC using BOPCl (46 mg, 1.2 equiv.). Purification according to Procedure Egave compound E-14.

Example W

Other compounds containing a thiophene-2-carboxamide moiety (such ascompounds E-1 or E-10) can be made by a convergent synthesis using theappropriate building blocks. The synthesis of compound E-10 is shown inScheme 26 as a representative example.

Compounds 260/261. Acid 245 (4.00 g, 1.0 equiv.) and4-(2-aminoethyl)morpholine (6.5 mL, 2.5 equiv.) were coupled accordingto Procedure B using HBTU (6.85 g, 1.1 equiv.). The reaction mixture waspoured into H₂O (300 mL) and extracted with Et₂O (4×). The combinedorganic layers were dried (MgSO₄) and evaporated to give compound 260 asa brown liquid. The material was treated with HCl-sat. AcOEt (200 mL),stirred at 0° C. for 1 h and treated with Et₂O (400 mL). The solvent wasdecanted and the remaining solids dried to give amine 261, which wasused without further purification.

Compound E-10. Trimer 12 (100 mg, 1.2 equiv.) and amine 261 (67.6 mg,1.0 equiv.) were coupled according to Procedure A using HBTU (89 mg,1.14 equiv.). Purification according to Procedure F gave compound E-10.

Example X

This example describes the synthesis of compounds containing animidazole moiety. The preparation of the dimeric imidazole intermediates274 and 276 is illustrated in Scheme 27.

Compounds 273/274. Acid chloride 270 (1.12 mL, 1.3 equiv.) and amine 272(1.50 g, 1.0 equiv.) were coupled according to Procedure D and purifiedaccording to Procedure G to give compound 273. Crude compound 273 wassaponified using Procedure H to give compound 274 (¹H-NMR). Compound 276was analogously prepared.

Compounds E-7, E-18, E-11, and E-12. Compound 274 was coupled to dimer211 to give compound E-7 and to dimer 13 to yield compound E-18, usingProcedure A and workup F. Similarly, compound 276 was coupled to thedimer 13 to yield compound E-11 and to the dimer 134 to yield compoundE-12, using Procedures A and F.

Example Y

This example describes the synthesis of compounds containing anisothiazole carboxamide moiety. The synthesis of compound E-2 is givenin Scheme 28 as a representative example.

Compound 281. Nitro acid 280 (300 mg, 1.2 equiv., Heindl et al., Eur. J.Med. Chem.—Chimica Therapeutica, 1975, 10, 591-593) and4-(2-aminoethyl)morpholine (0.188 mL, 1.0 equiv.) were coupled accordingto Procedure B. The mixture was diluted with H₂O (ca. 40 mL) andextracted with AcOEt (3×). The combined organic layers were dried(MgSO₄) and evaporated to give compound 281 (450 mg, ¹H-NMR).

Compound 282. Compound 281 (450 mg, crude product from above procedure)was hydrogenated using Procedure I to give compound 282 as a lightyellow powder (250 mg, ca. 90 to 95% pure by ¹H-NMR). This material wassubsequently used for the synthesis of compound E-2 without furtherpurification.

Compound E-2. Trimeric acid 87 (99 mg, 1.2 equiv.) and amine 282 (60 mg,1.0 equiv.) were coupled according to Procedure A using HATU (89 mg,1.14 equiv.). Workup according to Procedure F gave compound E-2.

Example Z

This example illustrates the synthesis of compounds containing an esterfunctionality at the C-terminus Scheme 29 depicts the syntheticmethodology for building blocks 292 and 294. Experimental details aregiven for building block 292, with building block 294 being preparedanalogously. Trimeric carboxylic acid 32 was subsequently coupled to theesters 292 and 294 using Procedure A to give compounds B-9 and B-10,respectively.

Compounds 291/292. A solution of 4-(2-hydroxyethyl)morpholine (3.4 mL,3.0 equiv.) in THF (20 mL) was treated at 0° C. portionwise with NaH(0.37 g, 1.0 equiv., 60% dispersion in mineral oil). The mixture wasstirred for 20 min until a clear solution was observed and treated witha solution of the ketone 290 (2.52 g, 1.0 equiv.) in THF (10 mL). Themixture was stirred for 3 h at 0° C., carefully diluted with sat.aqueous K₂CO₃ and AcOEt. The layers were separated and the aqueous phaseextracted with AcOEt (3×). The combined organic layers were dried(MgSO₄) and evaporated to give compound 291 (¹H-NMR, mass spectrum).Crude compound 291 was hydrogenated using Procedure I to give compound292, used without further purification.

Example AA

This example illustrates the synthesis of compounds containing a3-amino-5-carboxy isoxazole moiety, compounds E-3 to E-6, E-8 and E-19being representative. Schemes 30 and 31 detail the preparation ofcompounds E-3 and E-8 as models.

Compounds 311/312. Amine 310 [Lepage et al., FR 2,750,425 (1998)] (294mg, 1.88 mmol) and acid chloride 190 (231 μl, 1.88 mmol) were coupledaccording to Procedures D and G to give amide-ester 311 (550 mg).Amide-ester 311 (550 mg) was converted into acid 312 (500 mg) bysaponification according to Procedure I, except that the organic solventused was dioxane.

Compound E-3. Oxalyl chloride (131 μl, 1.5 mmol) was added to asuspension of acid 312 (31 mg, 0.12 mmol) in THF (1 mL). The reactionmixture was heated at 80° C. for 2 hours. All volatile components werethen removed under vacuum. A solution of amine 191 (162 μl, 1.2 Msolution in NMP, 0.194 mmol) in DMF (2 mL) and DIEA (0.5 mL) was thenadded and the reaction mixture was heated at 70° C. for 4 hours.Purification via Procedure E gave compound E-3.

Compound E-8. Oxalyl chloride (85 μl, 0.97 mmol) was added to asuspension of acid 312 (26 mg, 0.097 mmol) in THF (1 mL). The reactionmixture was heated at 80° C. for 2 hours. All volatile components werethen removed under vacuum. A solution of amine 13 (99 mg, 0.24 mmol) inDMF (2 mL) and DIEA (0.5 mL) was then added and the reaction mixture washeated at 70° C. for 4 hours. The reaction mixture was allowed to cooland was purified by Procedure E, giving compound E-8.

Example BB

Scheme 32 illustrates the synthesis of compounds having a2-amino-5-carboxy thiazole moiety adjacent to an N-terminal halogenatedbenzamide moiety, as represented by compound E-9.

Compounds 314/315. Amine 313 (Henichart et al., Heterocycles, v.32, No.4, p. 693, 1991) (200 mg, 1.16 mmol) and acid chloride 190 (142 μl, 1.16mmol) were coupled according to Procedure D. Workup according toProcedure G gave amide-ester 314 (360 mg). Amide-ester 314 (360 mg, 1.15mmol) was saponified to give amide-acid 315 (300 mg) using Procedure H,except that the organic solvent used was dioxane.

Compound E-9. Amide-acid 315 (100 mg, 0.35 mmol) and amine 13 (144 mg,0.35 mmol) were coupled according to Procedure C using BOPCl (89 mg,0.35 mmol). Purification according to Procedure E gave product E-9.

Example CC

This example illustrates the synthesis of compounds containing aninternal 4-amino-benzoic acid moiety. Representative examples arecompounds F-3 and F-4.

Compounds 317/318. Amine 191 (1.95 mL, 35% weight solution in NMP, 3mmol) and acid 319 (854 mg, 3.6 mmol) were coupled according toProcedure A using HBTU (1.25 g, 3.3 mmol). The reaction was poured intowater and the product extracted into AcOEt (3×). The combined organiclayers were dried (MgSO₄) and evaporated to give compound 317. ExcessTFA was added and the reaction mixture was stirred at RT to remove theBoc group. Following removal of volatile components under vacuum, theTFA salt of amine 318 was obtained, which was used without furtherpurification.

Compounds 320/321. Amine 80 (6.67 g, 35 mmol) and acid chloride 319(4.89 mL, 38.5 mmol) were coupled according to Procedure D. Product 320was obtained according to Procedure G. Crude product 320 was saponifiedusing Procedure H to give 321 (9.4 g) as a pure white solid.

Compound F-4. Acid 321 (167 mg, 0.6 mmol) and amine 318 (0.5 mmol) werecoupled according to Procedure C using BOPCl (140 mg, 0.55 mmol).Purification according to Procedure E gave the product F-4.

Example DD

This example illustrates the synthesis of more compounds containing a4-aminobenzoic acid moiety, as represented by compound F-6.

Compounds 322/323. Acid 316 (522 mg, 2.2 mmol) and4-(2-aminoethyl)morpholine (260 μl 2.0 mmol) were coupled according toProcedure C using BOPCl (535 mg, 2.1 mmol). The reaction mixture waspoured into water and the product extracted into ethyl acetate (3×). Thecombined organic layers were dried (MgSO₄) and evaporated to givecompound 322. Trifluoroacetic acid (excess) was added and the reactionmixture was stirred at RT to remove the Boc group. Following removal ofvolatile components under vacuum, the TFA salt of amine 323 wasobtained, which was used without further purification.

Compound F-19. Acid 12 (628 mg, 1.5 mmol) and amine 323 (1.0 mmol) werecoupled according to Procedure C using BOPCl (364 mg, 1.43 mmol).Purification according to Procedure E gave the product F-6.

Example EE

This example illustrates the synthesis of compounds containing a3-aminobenzoic acid moiety, as represented by compounds F-8, F-9 andF-10.

Compounds 325/326. Acid 324 (1.56 g, 6.6 mmol) and1-(2-aminoethyl)piperidine (845 μl, 6.0 mmol) were coupled according toProcedure A using HBTU (2.28 g, 6.0 mmol). The reaction mixture waspoured into water and the product extracted into ethyl acetate (3×). Thecombined organic layers were dried (MgSO₄) and evaporated to givecompound 325. Excess TFA was added and the reaction stirred at roomtemperature to remove the Boc group. Following removal of volatilecomponents under vacuum, the TFA salt of amine 326 was obtained and wasused without further purification.

Compound F-8. Acid 12 (653 mg, 1.56 mmol) and amine 326 (1.2 mmol) werecoupled according to Procedure C using BOPCl (321 mg, 1.26 mmol).Purification according to Procedure E gave the product F-8.

Example FF

This example illustrates the synthesis of compounds containing a2-amino-5-carboxypyridine moiety, as represented by compounds F-5 andF-7. Scheme 37 illustrates their synthesis by specific reference tocompound F-7.

Compounds 328/329. 6-Aminonicotinic acid (10 g, 72.4 mmol) in MeOH (55mL) was treated with a 4N solution of HCl in dioxane (54 mL, 217 mmol).The reaction mixture was heated at reflux for 12 hours. Removal ofvolatile components under vacuum left amino ester 327 as a white solid.Amino ester 327 (411 mg, 2.7 mmol) and acid chloride 5b (2.84 mmol) werecoupled using Procedures D and G to give product 328. Product 328 wassaponified using Procedure H to give acid 329.

Compound F-7. Acid 329 (103 mg, 0.35 mmol) and amine 13 (132 mg, 0.32mmol) were coupled according to Procedure C using BOPCl (85 mg, 0.33mmol). Purification according to Procedure E gave compound F-7.

Example GG

Compounds such as A-31 to A-33 and C-21 to C-25, in which the N-terminalphenyl group has a pendant amine substituent, from the correspondingfluoro polyamide by nucleophilic aromatic substitution of the fluorineby the corresponding amine, using reaction conditions of 48 hr at 60-70°C. in NMP. For instance, the treatment of compound A-3 with4-(2-aminoethyl)morpholine under such conditions gave compound A-31.

The foregoing detailed description of the invention includes passagesthat are chiefly or exclusively concerned with particular parts oraspects of the invention. It is to be understood that this is forclarity and convenience, that a particular feature may be relevant inmore than just the passage in which it is disclosed, and that thedisclosure herein includes all the appropriate combinations ofinformation found in the different passages. Similarly, although thevarious figures and descriptions herein relate to specific embodimentsof the invention, it is to be understood that where a specific featureis disclosed in the context of a particular figure or embodiment, suchfeature can also be used, to the extent appropriate, in the context ofanother figure or embodiment, in combination with another feature, or inthe invention in general.

Further, while the present invention has been particularly described interms of certain preferred embodiments, the invention is not limited tosuch preferred embodiments. Rather, the scope of the invention isdefined by the appended claims.

1-28. (canceled)
 29. A method of treating a bacterial infection in amammal, comprising administering to a patient in need of such treatmentan effective amount of a compound having the formula

wherein i is 3; one of X¹, X², and X³ is a ring vertex selected from thegroup consisting of —O—, —S—, and —NR²—, and the other two of X¹, X²,and X³ are ring vertices selected from the group consisting of ═N— and═CR⁴—, wherein each moiety

is the same or different and at least one moiety is other than

each R¹ is independently H, F, Cl, CN, CF₃, OH, N(R²)₂, OR² or asubstituted or unsubstituted (C₁-C₁₂)alkyl group, with the proviso thatat least one R¹ is F, Cl, CN or CF₃; each R² and R³ is independently Hor a substituted or unsubstituted (C₁-C₁₂)alkyl group; each R⁴ isindependently H, F, Cl, Br, I, CN, OH, NO₂, NH₂, or a substituted orunsubstituted (C₁-C₁₂)alkyl group; Z(R²)_(n) is

r is an integer ranging from 2 to 8, inclusive; each R²⁵ isindependently selected from the group consisting of H, CH₃, CH₂CH₃,CH₂CH₂CH₃, Or CH(CH₃)₂; said compound having at least one basic grouphaving a pK_(b) of 12 or less or a quaternized nitrogen group.
 30. Amethod according to claim 29, wherein

is selected from the group consisting of


31. A method according to claim 29, wherein the moiety

which is other than

is selected from the group consisting of


32. A method according to claim 29, wherein each R′ is independently H,F or Cl; i is 3; two of said moiety

are

and one is

and Z(R²)_(n) is


33. A method according to claim 29, wherein the compound has theformula:


34. A method according to claim 29, wherein the bacterial infection isan infection by Gram-positive bacteria.
 35. A method according to claim29, wherein the bacterial infection is an infection by drug resistantbacteria.
 36. A method according to claim 35, wherein the drug resistantbacteria is MRSA, MRSE, PRSP, or VRE.